Respiratory infection of neonatal mice with an aerosol of Bordetella pertussis provides a reproducible system for the study of the host-pathogen interactions involved in respiratory infection and subsequent disease. Analysis of genetic mutants of B. pertussis in this model is a powerful tool that allows us to identify new virulence determinants. Following infection with wild-type B. pertussis 18323, B. pertussis grows in the lungs of infected mice, concomitant with an increase in peripheral leukocyte count and decrease in weight, followed by death. Following aerosol infection with a transposon insertion mutant of 18323 deficient in the expression of pertussis toxin, this mutant persisted in the lungs of infected mice at lower levels than wild-type 18323 and did not cause leukocytosis, death or an inflammatory response. This data suggested that expression of pertussis toxin by B. pertussis is critical for high level persistence of this pathogen in the respiratory tract. A second TnphoA insertion mutant, determined to be activated by the environmentally responsive vir locus, lacks a major outer membrane protein, and is defective in bacterial persistence and induction of leukocytosis in this model; thus, we have identified a new virulence associated factor in this model. A second class of transposon insertion mutants, whose expression is repressed by the vir locus, was also analyzed in the mouse aerosol model. One of these mutants was determined to be defective in respiratory tract persistence, lymphocytosis, and the ability to kill, while a second vir repressed mutant remained as virulent as wild-type B. pertussis 18323. The role of these vir repressed genes in pathogenesis, remains to be elucidated.